PDBsum entry: 1gmo

Hormone

PDB id: 1gmo

Contents

Protein chains

(+ 2 more) 172 a.a. *

Ligands

IDS-SGN-IDS-SGN-IDS-SGN

SO4 ×8

EPE ×8

IDS-SGN-IDS-SGN-IDS-SGN-IDS

IDS-SGN-IDS-SGN-IDS-SGN-IDS-SGN

SGN-IDS-SGN-IDS-SGN-IDS-SGN-IDS-SGN

SGN-IDS-SGN-IDS-SGN-IDS ×2

Waters ×183

* Residue conservation analysis

PDB id:

1gmo

Name:

Hormone

Title:

Crystal structures of nk1-heparin complexes reveal the basis activity and enable engineering of potent agonists of the m receptor

Structure:

Hepatocyte growth factor. Chain: a, b, c, d, e, f, g, h. Fragment: nk1. Synonym: scatter factor, sf, hepatopoeitin a, hgf. Engineered: yes. Mutation: yes. Other_details: nk1 dimer in complex with heparin molecule

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: pichia pastoris. Expression_system_taxid: 644223.

Biol. unit:

Dimer (from PDB file)

Resolution:

3.00Å R-factor: 0.254 R-free: 0.295

Authors:

D.Lietha,D.Y.Chirgadze,B.Mulloy,T.L.Blundell,E.Gherardi

Key ref:

D.Lietha et al. (2001). Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor. EMBO J, 20, 5543-5555. PubMed id: 11597998 DOI: 10.1093/emboj/20.20.5543

Date:

20-Sep-01 Release date: 02-Oct-01

Protein chains

P14210  (HGF_HUMAN) -  Hepatocyte growth factor

Seq: 728 a.a.

Struc: 172 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1093/emboj/20.20.5543

EMBO J 20:5543-5555 (2001)

PubMed id: 11597998

Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor.

D.Lietha, D.Y.Chirgadze, B.Mulloy, T.L.Blundell, E.Gherardi.

ABSTRACT

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity.

Selected figure(s)

Figure 3.
Figure 3 Interaction of NK1 with heparin. (A) Stereoview of interactions between heparin monosaccharide units GlcN-5, IdoA-6, GlcN-7 and IdoA-8 with the N domain of NK1. The side chains of R73, K60 and T61 are shown along with conserved hydrogen bonds (in green). (B) Hydrogen bonding network and hydrophobic contacts between the N domain and heparin in crystal type A (left) and type B (right). Most hydrogen bonds occur in both crystal forms; crystal type A shows additional bonds from the side chains of Lys58 and Lys62. (C) The interface between the K domain of protomer F and heparin chain X. Side chains contributing to ionic interactions are shown. The interaction between K170 and heparin is not apparent in this structure, but is seen in several others.

Figure 4.
Figure 4 Heparin-induced oligomerization of wt- and mutant-NK1. (A) The mutations introduced in the N (HP11, HP12) and K domain (1K1, 1K2) of NK1. (B) Cross-linking of wt- and mutant-NK1 in the absence or presence of equimolar concentrations of 14mer heparin. Cross-linked proteins were analysed by western blotting and detected with an anti-HGF/SF polyclonal antibody (1W53). The position of molecular weight markers is also shown. (C -F) Gel filtration of wt- and mutant-NK1 in the absence or presence of equimolar concentrations of 14mer heparin. Chromatography was carried out on an HR30 Superdex-200 column equilibrated in PBS adjusted to 300 mM NaCl. The elution profile of the 1K1 mutant was identical to that of 1K2 and is not shown. wt-NK1 without heparin shows slight retardation in elution volume due to residual interaction with the column. Heparin induced a shift in the elution volume of wt-NK1 and of the K domain mutants but not of the N domain mutants HP11 and HP12.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2001, 20, 5543-5555) copyright 2001.

Figures were selected by an automated process.